1. IMMUNOLOGY B cell and humoral immunity Wei Chen, Associate Professor The Institute of Immunology Email: chenwei566@zju.edu.cnchenwei566@zju.edu.cn http://mypage.zju.edu.cn/566http://mypage.zju.edu.cn/566 Password: 8888

2. Study objective To understand the activation and function of B cells To be aware of the differentiation and maturation of B cells To know the development of B cells To distinguish the subsets and surface markers of B cells.

3. B Lymphocytes (B cells) B cells are an essential component of the adaptive immune system that play a large role in the humoral immune response. The principal functions of B cells are to make antibodies against antigens, perform the role of antigen- presenting cells (APCs) and eventually develop into memory B cells after activation by antigen interaction. The abbreviation "B", in B cell, comes from the bursa of Fabricius in birds, where they mature. In mammals, immature B cells are formed in the bone marrow and resides in LN and spleen.

4. B cells distribute in blood, lymphoid organs (lymph node, spleen, tonsil etc.) and mucosa. About 5-15% of the circulating lymphoid pool are B cells Naive mature B cells exit the bone marrow and migrate into the periphery. If these mature B cells encounter specific antigen, they become activated or tolerized. Following activation, Ag-specific B cells differentiate into antibody-forming cells (AFC) or memory B cells in the germinal centre. B Lymphocytes (B cells)

5. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

6. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

7. Development and migration of B cells (An overview)

8. The phase of B cell development

9. B lineage commitment HSC (hematopoietic stem cell) MPP (lymphoid/myeloid progenitor) ELP (earliest lymphocyte progenitor) ( 淋巴系髓系多能前体细胞 ) CLP (common lymphoid progenitor) ETP (early T-lineage progenitor)

10. B cell development related events 1. B cell development dependent on BM stromal cells 2. Transcription factors important for B lineage development 3. B cell development stages characterized by stage‐specific surface markers 4. B cell development is coupled with rearrangement of heavy chain and light chain 5. The role of Ig Heavy Chain and pre‐BCR 6. Immature B cells are tested for autoreactivity before they leave the bone marrow

12. 1. Regulators of Growth of Early B Lineage Cells FLT3-L ： induce IL7R IL-7: survival factor for T and B cells CXCL12(SDF-1): Retain precursors in BM Other cell-adhesion molecules VCM-1:binds to integrin VLA-4 SCF: interact with Kit

13. 2. B cell development is coupled with stage‐specific surface markers

14. 3. B cell development is coupled with gene rearrangement

15. Pre-BCR 与 BCR 结构示意图 前 B 细胞表面表达重链和替代轻链（ λ5 和 Vpre-B ），未 成熟 B 细胞表达完整的重链和轻链

16. 45 个 VH 23 个 DH 6 个 JH

18. The enzymes for gene rearrangement The products of the two genes Rag-1 and Rag-2 (recombination-activating genes) comprise the lymphoid-specific components of the recombinase. Tdt (terminal deoxynucleotidyl transferase) modify the ends of the broken DNA. TdT adds N-nucleotides to the V,D, and J exons, enabling the phenomenon of junctional diversity. Other enzymes: DNA exonuclease, DNA synthetas, DNA ligase.

19. B lineage commitment

20. The results of gene rearrangement

21. Allelic exclusion Isotype exclusion

22. Gene rearrangement of BCR Junctional diversity Somatic Hypermutation Affinity maturation in antibody responses The diversity of BCR

23. Gene rearrangement of TCR and BCR

24. Junctional diversity

25. Somatic Hypermutation

26. 4. Immature B cells are tested for autoreactivity before they leave the bone marrow----Negative Selection

27. 4. Immature B cells are tested for autoreactivity before they leave the bone marrow: Receptor Editing

28. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

29. BCR complexco-receptor CDs related to B cell activation

30. 1. BCR complex BCR (mIg): VH, VL----Ag binding site mature B cells: mIgM and mIgD. Function: specifically recognizes antigen. Ig  /Ig  (CD79a/CD79b): heterodimer cytoplasmic domains contain ITAM. Function: transduce the signals that lead to B cell activation.

31. CD19/CD21/CD81complex CD21=CR2, C3dR, EB virus receptor CD19/CD21/CD81 interactions with complement associated with antigen play a role in antigen-induced B-cell activation. 2. Co-receptors

32. The role of the coreceptor in B cell activation

33. (1)CD40 interacts with CD40L (Th cell) (2)CD80(B7.1), CD86(B7.2) Expressed on activated B cells and other APCs (3)ICAM-1 （ CD54 ）、 LFA-1 （ CD11α/CD18) ： mediate cell- cell interaction and co-stimulation 3. Co-stimulatory molecules

34. Other receptors CD20: function is unclear. It is suspected that it acts as a calcium channel in the cell membrane CD22 ： Inhibitory receptor with ITIM motif CD32 (FcγRII) ： Inhibitory receptor Cytokine receptors Complement Receptors Toll-like receptors MHC

35. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

36. Subtype of B cells 1. Conventional B cells (B-2 cells) 2. B-1 cells (expression of CD5)

37. B-2 cells : conventional B cells Recirculating follicular B cells : circulate between LN follicles and blood mIg: IgM, IgD Produce IgG after antigenic stimulation in the presence of T helper cells

38. B1 cells (CD5 + ): Many of the first B cells that appear during ontogeny express CD5, a marker originally found on T cells. (express mIgM, no mIgD). They respond well to TI-Ag and may also be involved in the Ag processing and presentation to T cells. Functions 1. produce anti-bacterial IgM the first line of defence against microorganisms; 2. produce polyreactive Ab clearance of denatured self components; 3. produce auto-Ab, thereby participating in the pathogenesis of some autoimmune diseases.

40. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

41. Functions of B cells 1. Production of antibody Abs prevent microorganism from entry into cells and eliminate microorganisms by opsonization causing phagocytosis, complement activation and toxin neutralization. 2. Ag presentation to T cells 3. Immune regulation Secretion of cytokines (TNF, IFN, IL-12) → M , DC, NK, B cell. Co-stimulation of T cells → T cell proliferation.

42. Phases of humoral immune responses

43. Functions of antibodies ADCC

44. Neutralization By Antiviral Antibodies

45. Downloaded from: StudentConsult (on 1 June 2006 04:16 PM) © 2005 Elsevier Antibody-mediated opsonization and phagocytosis of microbes

46. ADCC

47. Complement activation

48. Antigen presentation by B cells to T cells.

49. Mechanisms of Ig heavy chain class switching

50. Content Development of B cells B cell surface markers B cell subsets Functions of B cells humoral immunity

51. B cell‐mediated humoral immune response Humoral immunity is mediated by antibodies and is the arm of the adaptive immune response that functions to neutralize and eliminate extracellular microbes and microbial toxins. It is more important than cellular immunity in defending against microbes with capsules rich in polysaccharides and lipids. TD‐Ag ： T cell‐dependent TI‐Ag ： T cell‐independent

52. TD-Ag Most Ags in the nature mainly IgG Cell-mediated immune response Memory T and memory B cells TI-Ag Polysaccharides with repeated epitopes IgM no cell-mediated immunity, no memory requirement for Th cell help. The difference between TD-Ag and TI-Ag

53. B cell-mediated humoral immune response 1) Phases Antigen recognition phase proliferation and differentiation phase Effector phase 2) Types B2 → TD-Ag B1 → TI-Ag B cell-mediated immune response

54. Phases of humoral immune responses Effector phase

55. “First signal” a. BCR recognizes B cell epitopes b. Ig  /Ig  transfer signal c. coreceptors (CD21/CD19/CD81) Key point 1. B cells activated by Ag Immune response of B cells to TD-Ag B cell-mediated immune response

57. Antigen receptor-mediated signal transduction in B cells

59. Functional consequences of Ag-mediated B cell activation

60. 2. Th cell-mediated activation and differentiation of B cells Further activation Th cells → CD40L cytokines (IL-2, IFN-γ, IL-4, IL-5, IL-6, IL-13, etc.). Formation of germinal center Affinity maturation Heavy chain class switching Key point “Second signal” Site: GC (germinal center) B cell-mediated immune response

61. Ch. 11 p. 292

62. The interactions of Th cells and B cells in lymphoid tissues

63. The anatomy of humoral immune responses

64. Downloaded from: StudentConsult (on 1 June 2006 03:50 PM) © 2005 Elsevier Mechanisms of Th cell-mediated activation of B cells B cell-mediated immune response The edge of lymphoid follicle

65. Fully activation Th cells → CD40L cytokines Formation of germinal center Affinity maturation point mutations in the V regions Heavy chain class switching different Ab heavy chain classes Key point “Second signal” Site: GC (germinal center) 2. Th cell-mediated activation and differentiation of B cells

66. Germinal center Function: to generate B cells that produce antibodies with affinity maturation Germinal Center Reaction: Activated B cells give rise to Centroblasts （中心母细胞） - localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions Centroblasts give rise to Centrocytes （生发中心细胞） - migrate to the FDC-rich region of the Germinal Center - survival is dependent on interaction with FDC-bound Ag and presentation of Ag to Tfh cells - centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive Tfh cell help are selected and may differentiate into long-lived plasma cells or memory B cells

67. Germinal center

68. Ch. 11 p. 294

69. Fully activation Th cells → CD40L cytokines Formation of germinal center Affinity maturation point mutations in the V regions Heavy chain class switching different Ab heavy chain classes Key point “Second signal” Site: GC (germinal center) 2. Th cell-mediated activation and differentiation of B cells

70. The anatomy of humoral immune responses

71. Affinity maturation is the process by which the affinity of Abs produced in response to a protein Ag increases with prolonged and repeated exposure to that Ag. The increase in affinity is due to point mutations in the V regions, and particularly in the Ag-binding HVR, of the Abs produced. Affinity maturation occurs in the germinal centers of lymphoid follicles. Affinity maturation in Ab responses

72. Affinity maturation in antibody responses

73. Ch. 11 p. 294

74. Fully activation Th cells → CD40L cytokines Formation of germinal center Affinity maturation Heavy chain class switching is initiated by CD40L-mediated signals, and switching to different classes is stimulated by different cytokines Key point “Second signal” Site: GC (germinal center) 2. Th cell-mediated activation and differentiation of B cells B cell-mediated immune response

75. Isotype Switching Involves Recombination Between Specific Switch Signals

76. Ch. 11 p. 296

77. Effector phases of humoral immune responses Effector phase

78. 3. General features of Ab responses in vivo Key point Primary immune response - longer latent phase; - smaller peak response (lower Ab titer); - remaining in the serum at detectable levels for much shorter periods; - lower average affinity; - usually IgM; B cell-mediated immune response

79. Secondary immune response (The immune response followed by secondary antigenic response challenge) ‐ shorter latent phase; ‐ bigger peak response (higher Ab titer); ‐ remaining in the serum at detectable levels for much longer periods; ‐ higher average affinity; ‐ usually IgG.

80. Features of primary and secondary antibody responses

82. Immune response of B cells to TI-Ag B cell-mediated immune response Memory? * TI-1 (B cell mitogen) activate B cells * TI-2 activate mature B cells directly the repeated epitopes combine with BCR → BCR cross-linking → produce IgM No Th help, no memory, early effect, IgM

83. The mechanism of TI-Ag activating B1 cells (A) TI-1 Ag(B) TI-2 Ag B cell-mediated immune response

85. General concepts B cell activation: TD-Ag recoginition---First signal Th cell-mediated activation---Second signal Affinity maturation Heavy chain class switching General features of Ab responses in vivo B cell-mediated immune response

86. Movies for immune response The Immune Response: http://highered.mcgraw- hill.com/sites/0072507470/student_view0/chapter22/animation__the_i mmune_response.htmlhttp://highered.mcgraw- hill.com/sites/0072507470/student_view0/chapter22/animation__the_i mmune_response.html

87. Thank you!